Carotenoids are natural pigments widely existent in the natural world, and are polyene pigments having a color in the range of yellow to red or purple. Astaxanthin is one type of naturally-occurring carotenoids and exists in a free state or as an ester, or exists as various types of pigment proteins by bonding with proteins.
Astaxanthin is widely used as a coloring agent for fishes and chicken's eggs. Astaxanthin is also approved as a food additive and is widely used in fat and oil processed foods, protein foods, aqueous liquid foods and the like. Astaxanthin also has an anti-oxidation activity against peroxidation of a lipid induced by a free radical, a singlet oxygen quenching action which can be more effective by several hundred times than that of α-tocopherol or the like, and therefore is expected to be used as functional foods, cosmetic products and pharmaceutical drugs utilizing the strong anti-oxidation activity thereof.
Astaxanthin is distributed widely in the natural world in, for example, fishes such as salmon, trout and red sea bream; and crustaceans such as crab, shrimp and krill. Astaxanthin is also produced by bacteria belonging to genera Agrobacterium, Brevibacterium and Paracoccus as well as and microorganisms including Haematococcus green algae, Phaffia a yeasts and the like. Carotenoids such as astaxanthin, zeaxanthin or the like are industrially produced by a chemical synthesis method, however carotenoids derived from natural products are desired from a safety perspective.
In view of such a background, many methods for producing carotenoids containing astaxanthin derived from algae or microorganisms which are considered to be suitable for mass production have been reported.
For example, the following method for producing a carotenoid from a Haematococcus alga has been reported (Patent Document 1). A cystocyte of a cultured alga is treated with heated acetone to elute chlorophyll, i.e., a contaminant. Then, the cystocyte is spray-dried, and a carotenoid is extracted from the resultant dry cells with ethanol. However, a composition obtained by such a method still contains many contaminants from the organisms, and is not satisfactory in terms of 1) the carotenoid content, 2) the astaxanthin content, and the like.
In order to obtain a composition containing astaxanthin at a high content, the following method has been reported (Patent Document 2). A crude xanthophylls obtained according to the above-described method is allowed to react with lipase in the presence of water to decompose a neutral lipid, i.e., one of the contaminants, thereby separating the lipase enzyme-treated liquid into oil and water. From the separated oil layer, free fatty acid is separated from astaxanthin by distillation, whereby the astaxanthin is concentrated a purified. However, even after such complicated treating steps, a composition with an astaxanthin content of 30% or higher has not been obtained.
A method of obtaining astaxanthin contained at a ratio of 0.5 to 60% using a supercritical fluid extraction method (Patent Document 3) has also been reported. However, an astaxanthin fraction of a content less than the targeted content is produced as a sub-product during this treatment and discarded, or in order to increase the astaxanthin content of such a fraction, another concentration operation is required. Therefore, this production method is again not satisfactory, in terms of simplicity and cost, as an industrial method for producing a highly pure carotenoid containing a high content of astaxanthin with little contaminants derived from organisms.
As a method using Phaffia yeast, the following method has been reported (Patent Document 4). A fractured bacterial body of the yeast is treated with extraction using an organic solvent, and the oil-like crude extract obtained by concentrating the extract solution is purified by ion exchange chromatography, adsorption chromatography or the like to obtain astaxanthin. However, this method employs a plurality of column chromatographies to purify a crude solution having lower concentration astaxanthin and thus is difficult to be used for industrial application.
As another method, the following method has also been reported (Patent Document 5). A bacterial body obtained, by culturing Phaffia yeast is treated with extraction using acetone, and the resultant extract is concentrated to obtain a crude extract. A hydrocarbon-based solvent is added to this crude extract for crystallization. This method is highly simple, but the obtained composition contains a carotenoid at a content of about 70 to 73% (36 to 42% in terms of astaxanthin content). Accordingly, this method is not satisfactory as a method for producing a highly pure carotenoid with a small amount of contaminants derived from organisms. In addition, this method is also not satisfactory for the reason that there is a concern that acetone and the hydrocarbon-based solvent may remain in the carotenoid.
As methods using E-396 strain (FERM BP-4283: deposited on Apr. 27, 1993 (date of original deposition), International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, Higashi 1-1-1, Tsukuba-shi, Ibaraki-ken, Japan)), a bacterium that can produce astaxanthin, adonixanthin and the like, the following methods have been reported: a method employing extraction treatment by contacting an organic cyclic hydrophilic compound, which invokes a safety concern regarding the use in food production, with the bacterial body (Patent Document 6); a method employing supercritical fluid extraction like Patent Document 3 (Patent Document 7); a method employing liquid-liquid extraction by contacting the bacterial body with a water-soluble organic solvent, a nonpolar solvent and water (Patent Document 8); and a method employing extract on by contacting E-396 strain with a water-soluble organic solvent for concentration/crystallization and the washing the crystal with a solvent (Patent Document 9).
Under these circumstances, a method for industrially producing a highly pure carotenoid containing astaxanthin at a high content by a simple way without requiring any special facilities is strongly desired to be established.